A major part of diabetes therapy today involves treatment with intermediate and long-acting insulin products. These formulations are designed to control the patient's glucose levels during overnight time periods as well as provide one injection a day therapy in many patients.
A common feature of all these formulations is the fact that they are insoluble suspensions of insulin. Because of this, injection quantities can vary widely and glucose control after subcutaneous injection can be compromised (Skyler, J. S., Medical Clinics of North America, 72, 1337-1354 (1988)). Many of these formulations also require the addition of substantial amounts of protamine to provide long time action. Protamine is a fish protein which has been shown to cause antibody formation in some patients (Ellerhorst, J. A., et al., The American Journal of the Medical Sciences, 299, 298-301 (1987)).
with the advent of recombinant DNA technology, numerous analogs of insulin have been synthesized that can remain completely soluble in the formulation and yet have either quicker or more prolonged time action than natural insulin (Markussen, J., et al., Protein Engineering, 1, 215-223 (1987)). A most promising approach for the longer-acting insulin analogs is to formulate them to be completely soluble at a low pH (pH 3-4). After subcutaneous injection, the quick adjustment to the natural pH of the body environment (pH 7.4) causes these analogs to precipitate or crystallize. Their slow redissolution at pH 7.4 provides the time delay in action that is desired.
Two problems in this approach are as follow. First, the chronic administration of very acidic solutions may cause pain, skin necrosis and sloughing (DeLuca, P. P. and Rapp, R. P. Pharmaceutics and Pharmacy Practice; Banker, G. S. and Chalmers, R. K. Eds.; 238-278 (1982), J. B. Lippencott Co., Philadelphia, Pa.). Solutions closer to neutrality (pH 6-7) would clearly be more desirable in this regard. Second, since insulin analogs are unnatural to the body and as such may be recognized as non-self, antibodies to these insulin analogs may develop which can interfere with the patient's insulin therapy or cause other problems (Patterson, R., et al., Annals of Allergy, 64, 459-462 (1990)). Minimizing variations in a such a protein's structure could avoid this potential problem.
One insulin analog with intermediate time action and favorable solubility characteristics at pH 4-5 has already been described, namely di-arginine insulin (Zeuzem, S., et al., Diabetologia, 33, 65-71 (1990)). Di-arginine insulin however lacks one of the advantages of the present invention, that being solubility near pH 6. The present invention also shows superior time action characteristics over di-arginine insulin.
In addition to the physiological problem noted earlier with very acidic formulations, natural insulin-like molecules have another problem at low pH. Under acidic conditions, the asparagine residue in the number 21 position of the A-chain (A21) is very prone to deamidation and other side reactions that can lead to undesirable dimer and polymer formation (Markussen, J., et al., Protein Engineering, 2, 157-166 (1988)). These reactions proceed best below pH 4 and are almost nonexistent above pH 5 (Id.). Therefore, it would be most desirable in terms of human therapy to have an insulin-like molecule that possessed a prolonged hypoglycemic effect, low immunogenicity, and formulation solubility above pH 6.
This invention is based on the discovery that certain types of insulin analogs, herein referred to as tri-arginine (tri-arg) insulin and tri-arg insulin analogs, having the natural structure of insulin plus three additional arginine residues, have prolonged hypoglycemic activity in an animal model and can be formulated as a solution up to pH 6.1.